Neuropeptides play crucial roles in maintaining normal function of the brain and the brain's response to stresses, e.g. ischemia. Virtually all known neuropeptides are processed from larger precursors by the action of a set of processing enzymes in the secretory pathway. The biological function of precursor forms may differ profoundly from that of final processed forms to include even a switch from pro-apoptotic rather than anti-apoptotic function. Little is known about how the biosynthetic processing of neuropeptides may be affected by ischemic stress in the brain. Our preliminary studies indicate that ischemia causes attenuation in the activation of key neuropeptide processing enzymes and an accumulation of certain neuropeptides in precursor forms. Our hypothesis is: ischemia has adverse effects on the functional status of the neuropeptide processing system, thus impairing the ability of brain cells to produce certain protective peptide factors. This ischemia-induced attenuation of neuropeptide processing contributes to post-ischemia cell death in the brain. In the proposed study, we will first examine changes in the expression levels of several key neuropeptide processing enzymes and their enzymatic activities in ischemic rat brains. Then, we will determine the levels and molecular forms of neuropeptides that are known to have modulatory roles in the brain after ischemia and to require the action of processing enzymes investigated in this study for proper processing. Using in vitro ischemia models in cultured cells, we will investigate how ischemic stress may influence the activation and maturation processes of the processing enzymes and alter the production and secretion of neuropeptides. In parallel, we will analyze peptides secreted from ischemic cells using a quantitative proteomic approach. Finally, we will investigate if animals with specific deficiencies in neuropeptide processing may be more vulnerable to ischemic stress and determine if peptides secreted from processing-deficient neuronal cells may cause or exaggerate ischemic cells death. The proposed study will offer a novel mechanism concerning how ischemic stroke may damage the brain by attenuating neuropeptide processing. Our long-term goal is to elucidate the molecular mechanisms of neuropeptide processing-mediated stress response of the brain. [unreadable] [unreadable] [unreadable]